Covalent cross-linking of glutathione and carnosine to proteins by 4-oxo-2-nonenal.

Abstract

The lipid oxidation product 4-oxo-2-nonenal (ONE) derived from peroxidation of polyunsaturated fatty acids is a highly reactive protein cross-linking reagent. The major family of cross-links reflects conjugate addition of side chain nucleophiles such as sulfhydryl or imidazole groups to the C triple bond C of ONE to give either a 2- or 3-substituted 4-ketoaldehyde, which then undergoes Paal-Knorr condensation with the primary amine of protein lysine side chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or beta-alanylhistidine (carnosine), this would lead to circulating 4-ketoaldehydes that could then bind covalently to the protein Lys residues. This phenomenon was investigated by SDS-PAGE and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and LC-ESI-MS/MS with both tryptic and chymotryptic digestion). Under the reaction conditions of 0.25-2 mM ONE, 1 mM GSH or carnosine, 0.25 mM bovine beta-lactoglobulin (beta-LG), and 100 mM phosphate buffer (pH 7.4, 10% ethanol) for 24 h at 37 degrees C, virtually every Lys of beta-LG was found to be fractionally cross-linked to GSH. Cross-linking of Lys to carnosine was less efficient. Using cytochrome c and RNase A, we showed that ONE becomes more protein-reactive in the presence of GSH, whereas protein modification by 4-hydroxy-2-nonenal is inhibited by GSH. Stable antielectrophile-ONE-protein cross-links may serve as biomarkers of oxidative stress and may represent a novel mechanism of irreversible protein glutathionylation.

MALDI-TOF spectra of 0.25 mM β-LG modified by ONE at various concentrations (shown in figures) without GSH (A) or with 1 mM GSH (B). The mass difference of 118 Da in A is due to the Cys–ONE–Lys pyrrole cross-link, and 425 Da is due to the cross-link of GSH to the protein by ONE.

TIC (1st trace) of the tryptic digest of modified β-LG by ONE (d0:d9 = 1:1) in the presence of GSH and SIC and tandem mass spectrum of modified 76TKIPAVF83K by GSH.d0-ONE MA (2nd trace and 1st spectrum) or GSH.d9-ONE MA (3rd trace and 2nd spectrum).

Structure of AcGSH (A) and MALDI-TOF mass spectra of 0.25 mM of β-LG modified by 1.00 mM of ONE with or without 1.00 mM GSH or 1.00 mM AcGSH (B). The mass differences of 425 and 467 Da are due to the cross-link of GSH and AcGSH to the protein by ONE, respectively.

MALDI-TOF mass spectra of 0.25 mM of cytochrome c (A) or 0.25 mM of RNase A (B) modified by 1.00 mM of ONE without (a–e) or with (a'–e') 1.00 mM of GSH at various time. The mass difference of 154 Da is due to the Lys–ONE 4-ketoamide adduct or His–ONE MA and 425 Da is due to the cross-link of GSH to proteins.